Gastrointestinal Microbiota of Seabirds By Meagan Dewar Bachelor of Science, (Marine Biology) (BSc) Bachelor of Applied Biology (Hons) Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Deakin University 28th September, 2012 i “Earthworms are more important than humans. The fact is that earthworms are more important than people from an ecological perspective for the simple reason that earthworms can survive without people but people will not be able to survive without earthworms. People are dependent on earthworms; earthworms are not dependent upon humans. Worms can live on the planet without people; people can’t live on the planet without worms. Honeybees are more important than people, bacteria are more important than people – any species that is actually a foundation species which allows for the other species to survive is more important than the species up top. The fact is that the ecological law of diversity dictates that diversity of species is essential for the survival of each species within that diversity. Humans are equal to other species for the simple reason that if the other species were removed from the eco-systems that people inhabit, humans could not survive." Captain Paul Watson, Sea Shepherd ii ACKNOWLEDGEMENTS Over the past 4.5 years a lot of blood, sweat and tears have gone into achieving one thing, this thesis, and now that journey must come to an end. Throughout this time there have been numerous people who have helped me achieve my goal. First and foremost, I would like to thank my supervisors Dr Stuart Smith and Associate Professor John Arnould for this opportunity. I wish to thank Stuart for his encouragement, guidance, expertise and support over the past 4.5 years. Your assistance has been instrumental in achieving my goal. I wish to thank John for his advice, expertise and guidance. Your support has also been instrumental in helping me achieve my goal. I would also like to thank John Reynolds for your statistical assistance and advice. I would also like to thank my collaborators Dr Peter Dann (Phillip Island Nature Parks), Dr Phil Trathan (British Antarctic Survey) and Dr Rene Groscolas (Centre National de la Recherche Scientifique) for your logistical support, sample collection and editorial assistance. I would also like to thank the support crew from BAS; Stacey Adlard and Fabrice Le Bouard and CNRS; Vincent Viblanc, Nelly Mallosse, Goetz Eichhorn and René Groscolas for assisting with sample collection from Possession and Bird Island. I would also like to acknowledge my funding partners Holsworth Wildlife Research Endowment, Molecular Nutrition Group Strategic Research Centre (Deakin University), Phillip Island Nature Parks, Snowtown Valley, the French Polar Institute (IPEV) (project N° 119) and the British Antarctic Survey. Without your financial assistance this project would not have been possible. A special thank you to Dr Tiana Preston, Dr Louise Kelly, Joel Maloney, David, Kaye & Belinda Dewar for their assistance in the field at Phillip Island and to Nicole Schuman, Marcus Salton and Julie Baxter for their assistance in the field on Notch Island. Also to Tiana and Louise thank you for all the great penguin nights on the St Kilda breakwater and thank you for your support and advice throughout this time. To the Hutchesson family, Damien, Vanessa, Max and Oliver, thank you for your support, endless trips to the zoo, photography outings and the endless laughter. To my dearest friends Kathryn Trevaskis, Sarah Troia, Dimi Paraskevas, Suree Morrison and Marty Durkan I want to thank you for your love and support over the years and iii thank you for the many nights of gossip and laughter, but most of all thank you for your friendship. To my beautiful angels Bevan, Remy, Luca and Dylan, you have brought so much joy and happiness to my life. You always know how to make your Aunty Megs laugh, so thank you for making the hard times so much easier. To my family (Aunties, Uncles and Cousins), thank you for your support and believing in me over the years it has meant the world to me. To my sister, thank you believing in me, for your support, our wonderful holidays together and for putting up with me over the past 4.5 years. I know at times it hasn’t been easy. But thanks. To the Deakin crew Amy, Aimee, Sheena, Marissa, Paul and Lisa, thank you for sharing this journey with me. Thank you for your support, endless gossip sessions and daily coffee breaks. To my animals, Kitty, Claw, Pepper, Jessie, Sultan and Sebastian, you have been my source of comfort and joy over the past 23 years. I will never forget your endless love. Rest in Peace. To my new kitty, Nala, thank you for your love and cuddles these past few months. Finally, to the two most important people, my parents David and Kaye Dewar, without you this dream would never have become a reality. Thank you so very much for all the sacrifices you have made throughout my life to help me achieve my dreams. Thank you for your enduring love, support and belief in me. iv I dedicated this thesis to my parents: David and Kaye Dewar. Thank you for everything, without you this would not have been possible. v PUBLICATIONS FROM Ph.D. Dewar, M.L. Arnould, J.P.Y. Dann, P. Trathan, P. Groscolas, R and Smith, S.C. (2012). Inter-specific variations in the gastrointestinal microbiota of penguins. MicrobiologyOpen (Early View) http://onlinelibrary.wiley.com/doi/10.1002/mbo3.66/full FUTURE PAPERS FROM THESIS Dewar, M.L. Arnould, J.P.Y. Dann, P. Reynolds, J. Smith, S.C (2012) Variations in the Gastrointestinal Microbiota of Little Penguins (Eudyptula minor) During Development (In Preparation) Dewar, M.L. Arnould, J.P.Y. Trathan, P. Dann, P. Smith, S.C (2012) Influence of Fasting During Moult on the Gastrointestinal Microbiota of Penguins. (In Preparation) Dewar, M.L. Arnould, J.P.Y. Dann, P. Smith, S.C (2012) Inter-Specific Variations in the Gastrointestinal Microbiota of Procellariiformes (In Preparation) Dewar, M.L. Arnould, J.P.Y. Dann, P. Reynolds, J. Smith, S.C (2012) Variations in the Gastrointestinal Microbiota of Short-Tailed Shearwaters (Puffinus tenuirostris) During Development. (In Preparation) OTHER PUBLICATIONS Dewar, M.L., and Scarpaci, C. (2011). Microbial implications associated with stomach flushing of Little Penguins. The Victorian Naturalist 128 (4), 2011, 128–131 Saunders, S. Smith, S.C. Oppedisano, F. Dewar, M.L. Tang, M. (2012) Alteration of the intestinal microbiota by Lactobacillus rhamnosus GG and Peanut Oral Immunotherapy (P-POIT) in children with peanut allergy. Gut Pathogens (In preparation) Smith, S.C. Chalker, A. Dewar, M.L. Arnould, J.P.Y (2012). Age-related differences revealed in Australian fur seal Arctocephalus pusillus doriferus gut microbiota. FEMS Microbiology Ecology (In Review) vi COMMUNICATIONS Oral Communications of PhD results Dewar, M.L., Arnould, J.P.Y., Dann, P., Trathan, P., Smith, S.C. (2012). Variations in the Gastrointestinal Microbiota of Moulting and Non-Moulting Penguins. International Polar Year: Knowledge to Action. Montreal Canada 22-27 April 2012 Dewar, M.L., Arnould, J.P.Y., Trathan, P., Smith, S. (2011). Microbial Ecology of Penguins in Extreme Polar Regions. CAREX Life in Extreme Environments. 18-20th October 2011 Dewar, M.L, Arnould, J.P.Y., Trathan, P., Groscolas, R., Smith, S.C. (2010). Gastrointestinal microbiota of King Penguins (Aptenodytes patagonicus) and the Influence of geographical separation. 7th International Penguin Conference Abstracts. Boston, Massachusetts. 30 August – 3rd September 2010 Dewar, M.L., Arnould, J., Dann, P., Trathan, P., Groscolas, R., Smith, S.C. Exploring the gastrointestinal microbiota of king and little penguins using high throughput sequencing. (2010) 7th Annual Exercise and Nutrition Sciences Higher Degrees by Research Student Symposium, Deakin University Melbourne Australia. (Awarded best presentation) Poster Presentation of PhD results Dewar, M.L., Arnould, J.P.Y., Dann, P., Trathan, P., Groscolas, R., Smith, S. (2010). Changes in the gastrointestinal microbiota during fasting in penguins. 1st World Seabird Conference. Victoria, British Columbia, 7th -11th of September 2010 (Poster) Dewar, M.L., Arnould, J.P.Y., and Smith, S. (2009). Gastrointestinal Tract of Little penguins. Seabird Group 10th International Seabird Conference. Brugge, Belgium 27th -30th March 2009 (Poster). vii ABSTRACT The gastrointestinal (GI) tract contains a highly diverse and complex microbial ecosystem that consists of residential and transient microbes. At birth, the GI tract in avian species is devoid of microorganisms. Immediately after birth however, the GI microbiota is rapidly colonised by microbes. The GI microbiota plays a significant role in gut development and maturation, host biochemistry, physiology, metabolism, synthesis and secretion of essential vitamins, minerals and nutrients, energy extraction, fat metabolism and storage. To date, studies on the avian GI microbiota have primarily been focused on poultry, with a limited number of studies conducted on avian species living under natural conditions. This thesis aimed to address this knowledge gap through a study on the gastrointestinal microbiota of seabirds using faecal samples collected via cloacal swabs. The study was designed to characterise the gastrointestinal microbiota of marine seabirds under natural conditions and to examine the variation in microbial composition during development and to examine the influence of fasting during moult on the GI microbiota. The study species in this thesis included; king (Aptenodytes patagonicus), gentoo (Pygoscelis papua), macaroni (Eudyptes chrysolophus) and little penguin (Eudyptula minor), short-tailed shearwater (Ardenna tenuirostris), fairy prion (Pachyptila turtur) and common diving petrel (Pelecanoides urinatrix). DNA extracted from faecal samples was analysed using quantitative Real Time PCR (qPCR) and 16S rRNA pyrosequencing. Quantitative data obtained from the qPCR analysis highlighted significant differences in the abundance of Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria between all penguins (Chapter 2), Procellariiformes (Chapter 5), during development of little penguins (4) and in fasting penguins (study 4).